Antibodies against infectious diseases

ABSTRACT

Anti-CaENO1 antibodies and humanized antibodies are provided as an effective diagnostic agent or a therapeutic treatment against infections caused by  Candida  spp., preferably  Candida albicans, Candida tropicalis , fluconazole resistance  Candida  spp.,  Streptococcus, Staphylococcus.

FIELD OF THE INVENTION

The invention relates to an antibody against infectious diseases.Particularly, the invention provides an antibody against alpha-enolasefor diagnosis and treatment of against infections caused by Candida,fluconazole resistance Candida, Streptococcus, or Staphylococcus.

BACKGROUND OF THE INVENTION

Candida diseases are often chronic, difficult to treat, and carry a highmortality and morbidity despite anti-fungal therapy. Candida spp. arethe third leading cause of infections in ICUs globally, accounting forup to 90% of all fungal infections.

The diagnosis of invasive candidiasis is difficult due to the lack ofspecific clinical features and to the low sensitivity of blood culturefor isolation of Candida species, especially in patients receivingfluconazole prophylaxis. A positive blood culture for Candida spp.remains the gold standard for the diagnosis of candidemia. However,Candida spp. isolation may take too much time, thereby delayingeffective antifungal therapy. Candida albicans is the most importanthuman fungal pathogen. Particularly, Candida albicans (C. albicans) isan opportunistic human pathogen, which colonizes at several sitesincluding skin, oral tissue, gastrointestinal track and vagina. C.albicans is also a major pathogen responsible for 50.4% of clinicalcandidemia. Candidemia can occur when Candida yeasts enter thebloodstream and is rarely seen in healthy people. In recent decades, dueto the increase of patient population with defective immunologicalfunctions, Candidemia has become an important issue. Amphotericin (AmB)is a gold standard of antifungal treatment for fungi, but the severeside effect of this drug restricts its clinical application. Widespreadand prolonged use of azoles has led to the rapid development ofmultidrug resistance (MDR), which poses a major hurdle in antifungaltherapy. Several reports show that the incidence of resistance tofluconazole has risen during the last two decades.

Enolase is present in all tissues and organisms capable of glycolysis orfermentation. ENO1 was first identified as a key component of theglycolytic pathway. ENO1 is ubiquitously expressed in the cytosol andalso found on the cell surface as a plaminogen-binding receptor. Candidaalbicans ENO1 null mutants exhibit altered drug susceptibility, hyphalformation, and virulence. The expression of ENO1 in the fungal pathogenCandida albicans is critical for cell growth. Mutations on ENO1 inCandida albicans inhibit cell growth in the presence of glucose. ScFv isa recombinant antibody protein, which consists of the variable regionsof heavy chain (VH) and light chain (VL), combining by a linker peptide.In a previous study, anti-CaENO1 scFv antibody (CaS1) was isolated byphage display, but the interaction (epitope) of CaENO1 with CaS1 is notclear. There is a need to explore and develop a target regarding CaS1scFv inhibition against the interaction between CaENO1 and plasminogen.

SUMMARY OF THE INVENTION

The invention provides anti-CaENO1 antibodies (CaS1) and humanizedantibodies as effective diagnostic agent or therapeutic treatmentagainst infections caused by Candida (preferably Candida spp., morepreferably, Candida. albicans, Candida tropicalis,), fluconazoleresistance Candida (preferably fluconazole resistance Candida spp.),Streptococcus, or Staphylococcus.

In the invention, recombinant CaENO1 and CaS1 scFv were expressed andpurified successfully. CaS1 scFv recognizes ENO1 protein of C. albicans,S. pneumoniae, S. aureus; particularly, CaS1 scFv binds to fluconazoleresistance C. albicans and C. tropicalis from clinic and has weak crossreactivity to those of mouse and human.

The invention also provides an epitope sequence, comprising an aminoacid sequence consisting of ₂₈₃LYEQLISEYP₂₉₂ (SEQ ID NO:1),₂₇₈PQLADLYEQL₂₈₇ (SEQ ID NO:2), ₂₄₀KGKVGIAMDV₂₄₉ (SEQ ID NO:3) or₂₇₈PQLADLYEQLISEYP₂₉₂ (SEQ ID NO:4) located in CaENO1.

The invention also found that the polyclonal IgY antibodies showedbinding activity to the recombinant CaENO1 protein as well as nativeCaENO1 expressed by C. albicans, demonstrating that a strong humoralresponse was elicited in chicken. The complexity of antibody librariesconstructed with short or long linker was 2.4×10⁶ and 1.36×10⁷,respectively. After a stringent screening, a dominant CaS1 scFvspecifically recognized the ENO1 protein of C. albicans and C.tropicalis. In addition, CaS1 scFv binds to fluconazole resistance C.albicans and C. tropicalis from clinic. CaS1 also attenuated the growthof C. albicans and inhibited its adherence to oral epidermoid carcinomacells (OECM-1). In addition, CaS1 significantly inhibited the binding ofsurface ENO1 of C. albicans to plasminogen as showed by fibrinmatrix-gel degradation analysis. Noticeably, an in vivo animal testshowed that CaS1 antibody prolonged the survival time of mice withcandidemia. Consequently, the invention identifies a novel CaS1 scFvmonoclonal antibody with specific binding activity to CaENO1. All theresults together will provide great help in exploiting the therapeuticantibody drugs against the infection of C. albicans for clinicalapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) to (C) shows the expression and purification of recombinantCaENO1 protein. (A) Expression of recombinant CaENO1 protein was inducedby 0.1 (Lane 1), 0.5 (Lane 2), and 1.0 mM IPTG (Lane 3). The Coomassieblue was used to stain. (B) The mouse anti-His IgG and HRP conjugatedrabbit anti-mouse IgG were used in Western blot. Positive control:recombinant SaENO1 protein. (C) After expression and sonication, we usedNi⁺ sepharose to purify recombinant CaENO1 protein. Lane 1: supernatantof CaENO1 clone after Ni²⁺ sepharose binding. Lane 2: collection offirst time washing buffer. Lane 3: collection of first elution buffer.Lane 4: second time elution buffer. Lane 5: denoted Ni⁺ sepharose afterelution. The arrow denoted the recombinant CaENO1 protein molecularweight about 50 kDa.

FIG. 2 A and B shows analysis of anti-CaENO1 IgY antibodies using ELISAand Western blot. (A) The purified recombinant CaENO1 protein and BSA(negative control) were coated on ELISA plates, respectively. Then,blocking plates were incubated with a series diluted polyclonal IgYantibodies from pre-immunized or 7^(th)-immunized chicken. (B) The totalcell lysates of C. albicans were visualized on SDS-PAGE (Lane 1).Membranes were incubated with diluted polyclonal IgY antibodies frompre-immunized (lane 2) or 7^(th)-immunized (lane 3) chicken, followed byHRP-labeled donkey anti-chicken IgY (1:3,000). The detected ENO1 proteinexpressed in cell lysates of C. albicans is indicated by an arrow.

FIG. 3 shows sequence alignment of VH and VL domains of scFv antibodies.Nucleotide sequences of 10 scFv-S randomly selected from the antibodylibraries through 4th panning. The putative amino acid sequences werealigned with that of chicken germline gene. Sequence gaps wereintroduced to maximize the alignment by blank space.

FIG. 4 shows the expression and purification of CaS1 scFv. Afterexpression and sonication, Ni⁺ sepharose was used to purify CaS1 scFv.The Coomassie blue was used to stain. Lane 1: total cell lysate of CaS1scFv clone after expression. Lane 2: supernatant after Ni⁺ sepharosebinding. Lane 3: collection of first time washing buffer. Lane 4:collection of elution buffer. Lane 5: collection of second times elutionbuffer. Lane 6: Ni⁺ sepharose after elution. The arrow denoted CaS1 scFvmolecular weight about 25 kDa.

FIG. 5 (A) to (D) shows K_(D) determination of CaS1 scFv by ELISA andcompetitive ELISA. (A) Purified CaS1 scFv was used to recognizerecombinant CaENO1 protein. The CaS1 scFv were used as primary antibodywith series dilution. The goat anti-chicken light chain IgG was used assecondary antibody. The HRP conjugated donkey anti-goat IgG was used todecet. (B) OD value was calculated into percentage. K_(D) of CaS1 scFvis 0.498 ug/ml=1.88×10⁻⁸ M. (C) CaS1 scFv was used to recognize seriesdiluted free form recombinant CaENO1 protein that competed with fixedform recombinant CaENO1 protein. The goat anti-chicken light chain IgGwas used as secondary antibody. The HRP conjugated donkey anti-goat IgGwas used to detect. (D) We calculated OD value into percentage. K_(D) ofCaS1 scFv is 4.45 ug/ml=8.9×10⁻⁸ M. ELISA data were represented asmean±SD of the duplicated well.

FIG. 6 (A) to (D) shows binding activity of CaS1 scFv against ENO1proteins on Candida spp. by Western blot. (A) The total cell lysates of5 Candida spp. were visualized on SDS-PAGE (left). After transferredonto the NC membranes, they were probed with purified anti-CaENO1 from7th immunized chicken (1:3,000) (middle), or with CaS1 scFv (right) asdescribed in material and methods. Lanes 1-6 in (A) contained the totalcell lysates of C. albicans, C. krusei, C. tropicalis, C. parapsilosisand C. glabrate and purified recombinant CaENO1, respectively. (B) Totalcell lysate from fluconazole resistant (FLU^(R)) and fluconazolesusceptible (FLU^(S)) C. albicans (lanes 1-7 represent CA6-17, CA7-26,CA7-3, CA10-50, CA7-30, CA10-65, SC5314, respectively) by SDS-PAGE(left) and probed with CaS1 scFv by Western blot (right). (C) Total celllysate from FLU^(R) and FLU^(S) and C. tropicalis (lanes 1-5 representCT6-29, CT11-52, CT6-50, CT12-54, BCRC20520, respectively) by SDS-PAGE(left) and probed with CaS1 scFv by Western blot (right). (D) Thepurified ENO1 proteins of C. albicans, S. pneumonia, S. aureus, mouseand human were visualized on SDS-PAGE (left). After transferred onto theNC membranes, they were probed with purified CaS1 scFv (right) asdescribed. Lane M contained protein markers.

FIG. 7 (A) and (B) shows flow cytometry analysis of C. albicans withscFv CaS1. (A) C. albicans SC 5314 were cultured overnight and 10⁵ cellswere added to each tube with 2 ml YPD medium. Experiment antibodies wereadded to each tube (1) Anti-CaENO1 IgY (100 ug) (2) Control scFv (100ug) (3) scFv CaS1 (100 ug) (4) PBS, and cultured 2 hours at 37° C. in anincubator. Goat anti-chicken light chain (1:1500) antibody was used asdetected antibody; FITC donkey anti-goat antibody (1:1000) was used asdeveloped antibody to detect the reaction. Propidium iodide (PI) (1ug/ml) was used to detect cell death. (B) Quantification of flowcytometry result in the panel A (*, p<0.05;**, p<0.01). Flow cytometryis represented as mean±SD of the duplicated experiment.

FIG. 8 shows analysis of scFv CaS1 to C. albicans by immunofluorescencestaining. Anti-CaENO1 antibodies were used to detect alpha-enolaseprotein on C. albicans cell surface ENO1. (1) Detected with anti-CaENO1IgY and developed by FITC conjugation rabbit anti-chicken antibodies.(2) Detected with scFv CaS1 and developed by goat anti-chicken lightchain antibodies and FITC rabbit anti-goat antibodies. (3) An irrelevantscFv was used on negative control.

FIG. 9 shows the effects of CaS1 scFv on C. albicans growth and hyphalformation. (A) C. albicans were cultured in YPD medium overnight at 37°C. 10⁸ cfu/ml of C. albicans was mixed with equal volume of 0.5 mg/mlanti-CaENO1 IgY, 0.5 mg/ml CaS1 scFv or 1×PBS, respectively, at roomtemperature for 1 hr. 1 ul of each mixture in 10× dilutions was spottedon YPD agar plate and incubated at 37° C. overnight. (B) 10³ cfu of C.albicans was mixed with PBS (control), 10 or 100 □g/ml CaS1 scFv at roomtemperature for 1 hr. 1 ul of each mixture was spotted on YPD agar plateand incubated at 37° C. for 5 days. The hyphal formation was observedunder the microscope.

FIG. 10 shows CaS1 ScFv inhibited C. albicans adhesion to oral epidermalcells. C. albicans were mixed with 1×PBS, 50 ug or 100 ug CaS1 scFv at37° C. for 1 hr and added into OECM-1 cells in culture wells asdescribed in the text. After being washed, cells attached to C. albicanswere detected by adding HRP-conjugated anti-C. albicans antibodies.

FIG. 11 shows the effect of CaS1 scFv on the binding of ENO1 toplasminogen. The ability of CaS1 scFv to block the binding of CaENO1with plasminogen was evaluated by fibrin matrix-gel degradationanalysis. Various samples containing C. albicans only (1), C. albicans+1ug of plasminogen (2) or C. albicans+10 ug of plasminogen (3) werespotted on the plate. Similar experiments were carried out except C.albicans were mixed first with 10 ug (4) or 100 ug (5) of CaS1 scFv,followed by the addition of 10 ug of plasminogen. C. albicans+CaS1 scFvwithout plasminogen (6) were spotted as a negative control.

FIG. 12 (A) and (B) shows the expression and purification of hzCaS1 V1and V3 scFv. After expression and sonication, Ni⁺ sepharose was used topurify hzCaS1 V1 (A) and V3 (B) scFv. The Coomassie blue was used tostain (left panel). The mouse anti-HA IgG and HRP conjugated rabbitanti-mouse IgG in the Western blot (right panel). The arrow denotedhzCaS1 scFv molecular weight about 25 kDa. Lane 1: hzCaS1 scFv clonecell lysate. Lane 2: supernatant of hzCaS1 scFv after Ni²⁺ sepharosebinding. Lane 3: collection of first time washing buffer. Lane 4:collection of first elution buffer. Lane 5: Ni⁺ sepharose after elution.Positive control: CaS1 scFv.

FIG. 13 shows determination binding affinity CaENO1 with hzCaS1 V1, V3.The hzCaS1 V1, V3 and CaS1 scFv were used to recognize recombinantCaENO1 protein. (A) The Coomassie blue was used to stain. (B) The mouseanti-human κ, λ IgG and the HRP conjugated rabbit anti-mouse IgG wasused in the Western blot (right panel). (C) The mouse anti-HA IgG andHRP conjugated rabbit anti-mouse IgG was used. (D) The goat anti-chickenlight chain IgG and HRP conjugated donkey anti-goat IgG was used. Lane1: hzCaS1 scFv V1. Lane 2: hzCaS1 scFv V3. Lane 3: CaS1 scFv.

FIG. 14 (A) to (D) shows K_(D) determination of hzCaS1 V1 and V3 scFv byELISA. (A, C) Purified hzCaS1 V1 and V3 scFv were used to recognizerecombinant CaENO1 protein. The hzCaS1 V1 and V3 scFv were used asprimary antibody with series dilution. The goat anti-chicken light chainIgG was used as secondary antibody. The HRP conjugated donkey anti-goatIgG was used to decet. (B, D) OD value was calculated into percentage.The K_(D) or 50% effective concentration (EC₅₀) of scFv were calculatedand expressed by molarity (M). K_(D) of hzCaS1 V1 and V3 scFv is 1.51ug/ml=4.6×10⁻⁸ M and 2.12 ug/ml=8.4×10⁻⁸ M, respectivity. ELISA datawere represented as mean±SD of the duplicated well.

FIG. 15 shows that CaS1, hzCaS1 V1 and V3 scFv inhibits CaENO1 bindingto plasminogen. CaENO1 on Ni⁺ sepharose was treated with 100 ug hzCaS1V1, V3 and CaS1 scFv for 1 hour, following incubating with plasminogen(20 ug) for 1 hour. Each experimental group of CaENO1 with CaS1 scFvwere dropped onto gel and incubated at room temperature for 2 days toobserve gel degrade result. (1) plasminogen (1 ug/ul) only. (2) CaENO1on Ni Sepharose™ (10 ug). (3) CaENO1 on Sepharose™ (10 ug) withplasminogen (20 ug). (4-6) CaENO1 on Sepharose™ (10 ug) treated withhzCaS1 V1, V3, CaS1 scFv (100 ug) and incubated with plasminogen (20ug).

FIG. 16 shows that epitope of CaENO1 with CaS1 scFv is close to thebinding site of plasminogen with CaENO1. The epitope of CaENO1 with CaS1scFv (₂₄₀KGKVGIAMDV₂₄₉ (SEQ ID NO: 26) and ₂₇₈PQLADLYEQLISEYP₂₉₂ (SEQ IDNO: 27)) and plasminogen binding site (the last blocking regionindicated in the sequence of the figure) is indicated.

FIG. 17 shows the effect of CaS1 scFv on the survival of mice challengedwith C. albicans. The mice were grouped and challenged with the mixtureof 10⁶ of C. albicans containing 10 ug and 100 ug CaS1 scFv, 100 uganti-DA scFv (unrelated scFv control) or 1×PBS, respectively. Thesurvival of mice was monitored at 1 day intervals for 10 days. It isnoticeable that CaS1 scFv antibodies provides significant protectiveactivity against the lethal challenge of C. albicans in mice.

FIG. 18 shows Candida albicans biofilm formation inhibition assay.

DETAILED DESCRIPTION OF THE INVENTION

The invention develops an epitope of CaENO1 and anti-CaENO1 antibodies(CaS1) as an effective diagnostic agent or therapeutic treatment againstinfections caused by Candida, Streptococcus and Staphylococcus.

Definitions

In the description that follows, a number of terms are used for whichthe following definitions are provided to facilitate understanding ofthe claimed subject matter. Terms that are not expressly defined hereinare used in accordance with their plain and ordinary meanings.

Unless otherwise specified, “a” or “an” means “one or more.”

As used herein, the term “epitope” refers to the site on the antigen towhich an antibody binds.

As used herein, the term “candidiasis” refers to a fungal infection dueto any type of Candida (a type of yeast).

As used herein, the term “antibody” refers to single chain, two-chain,and multi-chain proteins and polypeptides belonging to the classes ofpolyclonal, monoclonal, chimeric, and humanized antibodies; it alsoincludes synthetic and genetically engineered variants of theseantibodies. “Antibody fragment” includes Fab, Fab′, F(ab′)₂, and Fvfragments, as well as any portion of an antibody having specificitytoward a desired target epitope or epitopes.

As used herein, the term “polyclonal antibody” refers to an antibodywhich is produced among or in the presence of one or more other,non-identical antibodies. In general, polyclonal antibodies are producedfrom a B-lymphocyte in the presence of several other B-lymphocytesproducing non-identical antibodies. Usually, polyclonal antibodies areobtained directly from an immunized animal.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies. Inother words, a monoclonal antibody consists of a homogeneous antibodyarising from the growth of a single cell clone (for example a hybridoma,a eukaryotic host cell transfected with a DNA molecule coding for thehomogeneous antibody or a prokaryotic host cell transfected with a DNAmolecule coding for the homogeneous antibody). These antibodies aredirected against a single epitope and are therefore highly specific.

As used herein, the term “single chain Fv” or “scFv” refers to anantibody in which the heavy chain and the light chain of a traditionaltwo chain antibody have been joined to form one chain. Typically, alinker peptide is inserted between the two chains to allow for properfolding and creation of an active binding site.

The term “linker peptide” includes reference to a peptide within anantibody binding fragment (e.g., Fv fragment) which serves to indirectlybond the variable heavy chain to the variable light chain. The linkermay be a series of a single amino acid or an alternating pattern ofamino acids, for example.

As used herein, the term “complementarity determining region” (CDR)refers to the non-contiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. CDRs havebeen described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977);Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofproteins of immunological interest” (1991); by Chothia et al., J. Mol.Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol.262:732-745 (1996), where the definitions include overlapping or subsetsof amino acid residues when compared against each other.

As used herein, the term “humanized antibody” refers to a recombinantprotein in which the CDRs from an antibody from one species; e.g., amurine or a chicken antibody, are transferred from the heavy and lightvariable chains of the antibody from the species into human heavy andlight variable domains (framework regions). The constant domains of theantibody molecule are derived from those of a human antibody. Thehumanized antibody may be achieved by various methods including (a)grafting only the non-human CDRs onto human framework and constantregions with or without retention of critical framework residues, or (b)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Suchmethods as are useful in practicing the present invention include thatdisclosed in Padlan, Mol. Immunol., 31(3):169-217 (1994).

As used herein, the term “chimeric antibody” refers to a recombinantprotein that contains the variable domains of both the heavy and lightantibody chains, including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody or a chicken antibody, more preferably a murine antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody.

As used herein, the term “phage display library” refers to a populationof bacteriophage, each of which contains a foreign cDNA recombinantlyfused in frame to a surface protein. The phage displays the foreignprotein encoded by the cDNA on its surface. After replication in abacterial host, typically E. coli, the phage which contain the foreigncDNA of interest are selected by the expression of the foreign proteinon the phage surface.

As used herein, the term “sequence identity” in the context of twonucleic acid or polypeptide sequences includes reference to thenucleotides (or residues) in the two sequences which are the same whenaligned for maximum correspondence over a specified comparison window.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA). An indication that two peptide sequences are substantiallysimilar is that one peptide is immunologically reactive with antibodiesraised against the second peptide. Thus, a peptide is substantiallysimilar to a second peptide, for example, where the two peptides differonly by a conservative substitution.

A “comparison window”, as used herein, includes reference to a segmentof about 10-20 residues in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison may be conducted by the local homology algorithm of Smith& Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignmentalgorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by thesearch for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci.USA 85:2444 (1988); by computerized implementations of these algorithms(including, but not limited to CLUSTAL in the PC/Gene program byIntelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), Madison, Wis., USA); the CLUSTAL program is well describedby Higgins & Sharp, Gene 73:237-244 (1988) and Higgins & Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucl. Acids Res. 16:10881-90 (1988);Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992);and Pearson, et al., Meth. in Molec. Biol. 24:307-31 (1994).

As used herein, the term “diagnostic” or “diagnosed” means identifyingthe presence or nature of a pathologic condition.

As used herein, the terms “treatment,” “treating,” and the like, coverany treatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

As interchangeably used herein, the terms “individual,” “subject,”“host,” and “patient,” refer to a mammal, including, but not limited to,murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

As used herein, the term “therapeutically effective amount” or“efficacious amount” refers to the amount of a subject anti-CaENO1antibody that, when administered to a mammal or other subject fortreating a disease, is sufficient to effect such treatment for thedisease.

As used herein, the term “biological sample” encompasses a variety ofsample types obtained from an individual, subject or patient that can beused in a diagnostic or monitoring assay. The definition encompassesblood and other liquid samples of biological origin, solid tissuesamples such as a biopsy specimen or tissue cultures or cells derivedtherefrom and the progeny thereof.

Epitopes Located in Candida albicans ENO1 (CaENO1) and AntibodiesAgainst Candida Alpha-Enolase

In one aspect, the invention provides an epitope sequence, comprising anamino acid sequence consisting of ₂₈₃LYEQLISEYP₂₉₂ (SEQ ID NO:1),₂₇₈PQLADLYEQL₂₈₇ (SEQ ID NO:2), ₂₄₀KGKVGIAMDV₂₄₉ (SEQ ID NO:3) or₂₇₈PQLADLYEQLISEYP₂₉₂ (SEQ ID NO:4) located in CaENO1. In oneembodiment, the epitope sequence comprises an amino acid sequenceconsisting of ₂₄₀KGKVGIAMDV₂₄₉ (SEQ ID NO:3) or ₂₇₈PQLADLYEQLISEYP₂₉₂(SEQ ID NO:4) located in CaENO1.

The purified CaS1 scFv is used to recognize recombinant CaENO1 protein.The epitope region is mapped to contain 198 bp nucleotides, whichdeduces the following amino acid sequence (residues 235 to 300):DKAGYKGKVGIAMDVASSEFYKDGKYDLDFKNPESDPSKWLSGPQLADLYEQLISEYPIVS IEDPF (SEQID NO: 19) (66 amino acids). To further determine the epitopic location,site directed mutagenesis is used to construct nine peptide-expressingphages according to the nucleotide sequences of 198 bp of mappedantigenic fragment. In one embodiment, CaS1 scFv antibody binds to afragment of plasminogen spanning amino acid residues 301 to 437, whichsequences areAEDDWDAWVHFFERVGDKIQIVGDDLTVTNPTRIKTAIEKKAANALLLKVNQIGTLTESIQAANDSYAAGWGVMVSHRSGETEDTFIADLSVGLRSGQIKTGAPARSERLAKLNQILRIEEELGSEAIYAGKDFQKA (SEQ ID NO:20).

In one aspect, the present invention provides an isolated anti-CaENO1antibody or an antigen-binding portion thereof, comprising at least oneof a light chain complementarity determining region 1 (L-CDR1) of SEQ IDNO:5 or a variant having amino acid sequence with at least 80% identityto any of L-CDR1s; a light chain CDR2 (L-CDR2) of SEQ ID NO:6 or avariant having amino acid sequence with at least 80% identity to any ofL-CDR2s; and a light chain CDR3 (L-CDR3) of SEQ ID NO:7 or a varianthaving amino acid sequence with at least 80% identity to any of L-CDR3s;and at least one of a heavy chain CDR1 (H-CDR1) of SEQ ID NO:8 or avariant having amino acid sequence with at least 80% identity to any ofH-CDR1s; a heavy chain CDR2 (H-CDR2) of SEQ ID NO:9 or a variant havingamino acid sequence with at least 80% identity to any of H-CDR2s; and aheavy chain CDR3 (H-CDR3) of SEQ ID NO: 10 or a variant having aminoacid sequence with at least 80% identity to any of H-CDR3s; such thatsaid isolated antibody or antigen-binding portion thereof binds toCaENO1. Preferably, the sequence identity as mentioned above is at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99%.

The amino acid sequences of the complementarity determining regions(CDRs) in heavy chains and light chains are listed in the table below.

CDRs of Light Chain L-CDR1 L-CDR2 L-CDR3 SGSYG SNN GSRDSSYVGV(SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7)

CDRs of Heavy Chain H-CDR1 H-CDR2 H-CDR3 GFTFIDYG IGSSGSSTAKSAGGYCVNGAGCNGGSIDA (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)

The preceding CDR sequences are determined by using the internationalImMunoGeneTics information System® (www.imgt.org).

In some embodiments, the isolated anti-CaENO1 antibody is a monocloncalantibody, chimeric antibody, humanized antibody or human antibody.

Accordingly, the invention provides a light chain of an anti-CaENO1 scFvmonoclonal antibody (CaS1) comprising an amino acid sequence consistingof the sequence:ALTQPSSVSANLGGTVKITCSGGSGSYGWYQQKSPGSAPVTVIYSNNQRPSNIPSRFSGSPSGSTGTLTITGVQADDEAVYFCGSRDSSYVGVFGAGTTLTVL (SEQ ID NO:11). The inventionprovides a heavy chain of an anti-CaENO1 scFv monoclonal antibody (CaS1)comprising an amino acid sequence consisting of the sequence:TVTLDESGGGLQTPRGALSLVCKASGFTFIDYGMQWVRQAPGKGLEWVAGIGSSGSSTNYGAAVKGRATISRDDGQSTVRLQLNNLRAEDTGTYYCAKSAGGYCVNGAGCNGGSIDAWG HGTEVIVSS(SEQ ID NO:12). The invention also provides an anti-CaENO1 scFvmonoclonal antibody (CaS1) comprising a light chain having an amino acidsequence consisting of the sequence of SEQ ID NO: 11 and a heavy chaincomprising an amino acid sequence consisting of the sequence SEQ IDNO:12.

The antibody molecule can be a polyclonal or a monoclonal antibody orany other suitable type of an antibody, such as a fragment or aderivative of an antibody, a single chain variable fragment (ScFv) or asynthetic homolog of the antibody, provided that the antibody has thesame binding characteristics as, or that has binding characteristicscomparable to, those of the whole antibody. In some embodiments, theantibody can be recombinantly produced, e.g., produced by any suitablephage display or combinatorial methods. Various phage display andcombinatorial methods for generating antibodies are known in the art (asdescribed in, e.g., Griffths et al. (1993) EMBO J 12:725-734; Hawkins etal. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982).

Antibody fragments can be produced by cleaving a whole antibody, or byexpressing DNA that encodes the fragment. Fragments of antibodies may beprepared by methods described in the published literature (Lamoyi etal., J. Immunol. Methods, 56:235, 1983; Parham, J. Immunol., 131:2895,1983). Such fragments may contain one or both of an Fab fragment and anF(ab′)2 fragment. Such fragments may also contain single chain variablefragment antibodies, i.e. scFv, dibodies, or other antibody fragments.

Single chain variable fragments (scFv) are polypeptides that consist ofthe variable region of a heavy chain of an antibody linked to thevariable region of a light chain with a short peptide linker). Thus, thescFv comprises the entire antibody-combining site. These chains may beproduced in bacteria, or in eukaryotic cells.

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding variable light chain and variable heavy chainsequences for an antibody of interest may be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. The variable heavy or light chain sequence genes of anantibody from a cell that expresses a murine antibody can be cloned byPCR amplification and sequencing. To confirm their authenticity, thecloned V_(L) and V_(H) genes can be expressed in cell culture as achimeric antibody as described by Orlandi et al., (Proc. Natl. Acad.Sci., USA, 86: 3833 (1989)). Based on the variable heavy or light chaingene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

In one embodiment, the invention provides the followings amino acids ofthe light chains and heavy chains of humanized antibodies.

Embodiments of Amino Acid Sequences of Light ChainsDIQLTQSPSSLSASVGDRVTITCRASSGSYSLGWYQQKPGKAPKRLIYSNNSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCGSRDSSYVGVFGQGTKVEIK (SEQ ID NO: 13) (hzCaS1-V1 scFv)DIQMTQSPSSLSASVGDRVTITCRASSGSYGVAWYQQKPGKAPKWYSNNFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGSRDSSYVGVFGQGTKVEIK (SEQ ID NO: 14) (hzCaS1-V3 scFv)Embodiments of Amino Acid Sequences of Heavy chainsEVKLEESGGGLVQPGGSMKLSCAASGFTFIDYGMDWVRQSPEKGLEWVAEIGSSGSSTHYAESVKGRFTVSRDDSKSSVYLQMNSLRAEDTGIYYCAKSAGGYCVNGAGCNGGSIDSAWGQGTLVTVSA (SEQ ID NO: 15) (hzCaS1-V1 scFv)EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYGIHWVRQAPGKGLEWVAGIGSSGSSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAKSAGGYCVNGAGCNGGSIDAWGQGTLVTVSS (SEQ ID NO:16) (hzCaS1-V3 scFv)

In some embodiments, the invention provides a light chain comprising anamino acid sequence having a sequence selected from the group consistingof those as set forth in SEQ ID NOs:13 to 14.

In some embodiments, the invention provides a heavy chain comprising anamino acid sequence having a sequence selected from the group consistingof those as set forth in SEQ ID NOs:15 to 16.

In further embodiments, the invention comprises a humanized antibody,comprising (i) a light chain having an amino acid sequence as set forthin the sequence selected from the group consisting of SEQ ID NOs:13 to14 or a variant having at least 80% identity to any of SEQ ID NOs:13 to14, and (ii) a heavy chain having an amino acid sequence as set forth inthe sequence selected from the group consisting of SEQ ID NOs: 15 to 16or a variant having at least 80% identity to any of SEQ ID NOs:15 to 16.Preferably, the sequence identity as mentioned above is at least 90%,91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In a preferred embodiment, the humanized antibody comprises (i) a lightchain having an amino acid sequence as set forth in SEQ ID NO:13 and(ii) a heavy chain having an amino acid sequence as set forth in SEQ IDNO: 15 (hzCaS1-V1). In another preferred embodiment, the humanizedantibody comprises (i) a light chain having an amino acid sequence asset forth in SEQ ID NO:14 and (ii) a heavy chain having an amino acidsequence as set forth in SEQ ID NO:16 (hzCaS1-V3).

In addition to recombinant methods, the antibodies and variants thereofthat are disclosed herein can also be constructed in whole or in partusing standard peptide synthesis. Solid phase synthesis of thepolypeptides can be accomplished by attaching the C-terminal amino acidof the sequence to an insoluble support followed by sequential additionof the remaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.

Compositions and Methods of Administrations

Certain embodiments relate to a pharmaceutical composition comprising anepitope of the invention or an antibody against Candida alpha-enolase ofthe invention and a pharmaceutically acceptable carrier or excipient. By“pharmaceutically acceptable carrier” is intended, but not limitedthereto, a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type known topersons skilled in the art. Diluents, such as polyols, polyethyleneglycol and dextrans, may be used to increase the biological half-life ofthe conjugate.

The pharmaceutical compositions of the present invention can beformulated according to conventional methods (for example, Remington'sPharmaceutical Science, latest edition, Mark Publishing Company, Easton,U.S.A.), and may also contain pharmaceutically acceptable carriers andadditives. Examples include, but are not limited to, surfactants,excipients, coloring agents, flavoring agents, preservatives,stabilizers, buffers, suspension agents, isotonic agents, binders,disintegrants, lubricants, fluidity promoting agents, and corrigents,and other commonly used carriers can be suitably used. Specific examplesof the carriers include light anhydrous silicic acid, lactose,crystalline cellulose, mannitol, starch, carmellose calcium, carmellosesodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose,polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin,medium-chain triglyceride, polyoxyethylene hardened castor oil 60,saccharose, carboxymethyl cellulose, corn starch, inorganic salt, andsuch.

The invention also provide methods for inhibition of Candida,Streptococcus and Staphylococcus growth and treatment of infectioncaused thereby.

One embodiment is directed to a method for inhibition of Candida,Streptococcus or Staphylococcus growth and treatment of infection causedthereby in a subject, comprising administering an antibody againstalpha-enolase of the invention to the subject. Accordingly, alsoprovided is the use of an antibody against alpha-enolase of theinvention in the manufacture of a medicament for inhibition of Candida,Streptococcus or Staphylococcus growth or treatment of infection causedthereby in a subject.

Another embodiment is directed to a method for prevention of growth orinfection caused by Candida, Streptococcus or Staphylococcus in asubject, comprising administering an epitope of the invention to thesubject. Accordingly, also provided is the use of epitope of theinvention in the manufacture of a medicament for prevention of growth orinfection caused by Candida, Streptococcus or Staphylococcus in asubject.

In some embodiments, the Candida is C. albicans, Candida tropicalis, andCandida glabrate, C. albicans, the Streptococcus is S. pneumoniae andthe Staphylococcus is S. aureus.

Another embodiment is directed to a method for treating candidiasis in asubject comprising administering an antibody against alpha-enolase ofthe invention to the subject. Accordingly, also provided is the use ofan antibody against alpha-enolase in the invention in the manufacture ofa medicament for treatment of candidiasis in a subject. Preferably, thecandidiasis disease is invasive candidiasis, antibiotic candidiasis,dysbiosis of the gut mycobiota, onychomycosis, cutaneous candidiasis, ormucosal candidiasis.

Another embodiment is directed to a method for inhibition offluconazole-resistance Candida growth or treatment of infection causedthereby in a subject, comprising administering an antibody againstalpha-enolase of the invention to the subject. Accordingly, alsoprovided is the use of an antibody against alpha-enolase in theinvention in the manufacture of a medicament for inhibition offluconazole-resistance Candida spp. growth or treatment of infectioncaused thereby in a subject. Preferably, the fluconazole-resistanceCandida is Candida spp.; more preferably, the fluconazole-resistanceCandida is fluconazole resistance C. albicans or C. tropicalis.

Another further embodiment is to provide a method for inhibition ofbiofilm formation caused by Candida, comprising administering anantibody against alpha-enolase of the invention to the subject.Accordingly, also provided is the use of an antibody againstalpha-enolase in the invention in the manufacture of a medicament forinhibition of biofilm formation caused by Candida. Preferably, theCandida is C. albicans, Candida tropicalis and Candida glabrate, C.albicans. Biofilms are one of the main reasons why a Candida overgrowthis so hard to defeat. A longstanding Candida overgrowth has had plentyof time to create biofilms, and these are very resistant to manytreatments. The longer the biofilm has had to develop, the moreresistant it will be to antifungal treatments. This is why usingantifungal drugs alone is often not enough to inhibit a Candidaovergrowth. Surprisingly, the invention found that the antibodies of theinvention can effectively inhibit Candida biofilm formation.

The above methods also comprise administering the antibody againstalpha-enolase of the invention concomitantly with or subsequent to otherstandard therapies as described in updated guidelines published in March2009 by the Infectious Disease Society of America (IDSA) (Pappas P G,Kauffman C A, Andes D, Benjamin D K Jr, Calandra T F, Edwards J E Jr, etal. Clinical practice guidelines for the management of candidiasis: 2009update by the Infectious Diseases Society of America. Clin Infect Dis.2009 Mar. 1. 48(5):503-35).

In preferred embodiments, the subject is a mammal. Exemplary mammalsinclude human, pig, sheep, goat, horse, mouse, dog, cat, cow, etc.Diseases that may be treated with the antibody or a pharmaceuticalcomposition thereof include candidiasis. Examples of candidiasis includebut are not limited to invasive candidiasis, antibiotic candidiasis,dysbiosis of the gut mycobiota, onychomycosis, cutaneous candidiasis,and mucosal candidiasis.

The antibody against alpha-enolase as disclosed herein or thepharmaceutical composition thereof may be administered intravenously,topically, intra-peritoneally, intra-arterially, intra-thecally,intra-vesically, or intratumorally. One of ordinary skill willappreciate that effective amounts of the antibody against alpha-enolaseor its composition can be determined empirically. It will be understoodthat, when administered to a human patient, the total daily usage of theantibody against alpha-enolase or its composition will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors: the type and degree of thecellular response to be achieved; activity of the specific the antibodyagainst alpha-enolase or its composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the antibody againstalpha-enolase or its composition; the duration of the treatment; drugsused in combination or coincidental with the antibody againstalpha-enolase or its composition; and like factors well known in themedical arts.

Each of the above identified compositions and methods of treatment mayadditionally include an additional anti-Candida, anti-Streptococcus oranti-Staphylococcus drug. In some embodiments, anti-Candida drugssuitable for use with the present invention include, but are not limitedto, fluconazole, itraconazole, posaconazole, echinocandins caspofungin,micafungin, anidulafungin, voriconazole, lipid formulations ofamphotericin B, Ketoconazole, clotrimazole, econazole, ciclopirox,miconazole, ketoconazole and nystatin. In the method of treatment, theantibody against alpha-enolase of the invention can be administeredconcurrently, subsequently or separately with the additional one or moreanti-Candida, anti-Streptococcus or anti-Staphylococcus drugs.

Diagnosis of Candida, Streptococcus or Staphylococcus Infection

The present invention surprisingly found that anti-CaENO1 antibodies(CaS1) are an effective diagnostic agent or therapeutic treatmentagainst Candida, Streptococcus or Staphylococcus infection. Accordingly,in another aspect, the present invention provides a method fordiagnosing Candida, Streptococcus or Staphylococcus infection in abiological sample of a subject, comprising contacting an anti-CaENO1antibody of the invention with the biological sample and detecting thebinding of the anti-CaENO1 antibody to an epitope of CaENO1 of theinvention wherein the presence of the binding indicates that the subjectis suspected of suffering from Candida, Streptococcus or Staphylococcusinfection. Alternatively, the present invention provides a method fordiagnosing Candida, Streptococcus or Staphylococcus infection in abiological sample of a subject, comprising contacting an epitopesequence of the invention with the biological sample and detecting thebinding of the epitope sequence of the invention to an anti-CaENO1antibody wherein the presence of the binding indicates that the subjectis suspected of suffering from Candida infection.

In the present invention, detection includes quantitative andqualitative detection. Examples of qualitative detection include thefollowing: simple detection of the presence or absence of the binding ofthe anti-CaENO1 antibody to an epitope of CaENO1 of the invention;determination of whether or not the binding is present above a certainamount; and comparison of the amount of the binding with that of othersamples (for example, a control sample).

Biological samples used in the diagnostic methods of the presentinvention are not particularly limited as long as they are samples thatmay contain a CaENO1 protein. Specifically, samples collected from thebody of an organism such as a mammal are preferred. Samples collectedfrom humans are more preferred. Specific examples of the test samplesinclude blood, interstitial fluid, plasma, cerebrospinal fluid, synovialfluid, pleural fluid, serum, lymphatic fluid, saliva, urine, tissue andascites.

In the present invention, “control” refers to samples serving as astandard for comparison, including negative controls and biologicalsamples from healthy subjects. Negative control can be obtained bycollecting biological samples from healthy subjects and mixing them asnecessary. The level of binding of the anti-CaENO1 antibody to anepitope of CaENO1 in a control can be detected in parallel with thebinding level in the biological sample of a subject. Alternatively, bydetecting the binding level in biological samples of many healthysubjects in advance, a standard expression level in healthy subjects canbe determined statistically.

In the present invention, the binding level can be determined by anymethod. Methods for detecting the binding in a test sample are notparticularly limited. An immunological method using an anti-CaENO1antibody for detection is provided, such as radioimmunoassay (RIA);enzyme immunoassay (EIA); fluorescence immunoassay (FIA); luminescenceimmunoassay (LIA); immunoprecipitation (IP); turbidimetric immunoassay(TIA); Western blotting (WB); immunohistochemical (IHC) method; andsingle radial immunodiffusion (SRID).

The present invention also provides diagnostic agents or kits fordiagnosing a Candida, Streptococcus or Staphylococcus infection,comprising a diagnostic agent for detecting the binding of theanti-CaENO1 antibody to a CaENO1 in a test sample. The diagnostic agentsof the present invention comprise at least a Candida infection.

Kits for diagnosing cancer can be produced by combining the agents fordiagnosing a Candida infection with another element used for detectingthe anti-CaENO1 antibody. More specifically, the present inventionrelates to kits for diagnosing a Candida, Streptococcus orStaphylococcus infection which comprise an anti-CaENO1 antibody thatbinds to CaENO1 and a reagent for detecting binding between the antibodyand CaENO1. In addition, instructions that describe the measurementoperation can be attached to the kits of the present invention.

EXAMPLES

Materials and Methods

Expression and Purification of his-CaENO1 Protein

Briefly, C. albicans alpha-enolase genes were constructed in pQE30plasmids to form pQE30-CaENO1 vector, and then the resulting vectorswere transformed with E. coli BL21 cells. The bacterial culture wasgrown in 10 ml LB medium containing ampicillin (50 μg/ml) at 37° C.overnight, diluted 10-fold in the same LB medium and further grown untilthe OD₆₀₀ reached between 0.6 and 1.0. To induce CaENO1 proteinexpression, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to afinal concentration of 0.5 mM in the culture. The cell pellet wasresuspended in 2 ml of 1×PBS containing 1% Triton x-100 and lysed bythree cycles of freezing (−70° C.) and thawing (37° C.). Aftercentrifugation, the resulting cellular lysate was incubated with aNi²⁺-charged resin column to purify His-CaENO1 protein according to themanufacturer's instruction (GE Healthcare Bio-Sciences AB, Sweden). ENO1from human, mouse, S. pneumonia, S. aureus were expressed and purifiedin the same way.

Animal Immunization

Female white leghorn (Gallus domesticus) chickens were immunized with 50□g of purified His-CaENO1 in an equal volume of Freund's completeadjuvant (Sigma, USA) by intramuscular injection. Three additionalimmunizations with His-CaENO1 in Freund's incomplete adjuvant wereperformed at intervals of 7 days. After each immunization, polyclonalIgY antibodies in egg yolk were partially purified and titrated by anenzyme-linked immunosorbent assay (ELISA) to determine the presence ofhumoral anti-his-CaENO1 immune response. The IgY antibodies werepurified from the yolk separated from the egg white using 10% Dextransulphate as described previously (Akita E M, Nakai S. Comparison of fourpurification methods for the production of immunoglobulins from eggslaid by hens immunized with an enterotoxigenic E. coli strain. J ImmunolMethods 1993; 160: 207-14; Akita E M, Nakai S. Production andpurification of Fab′ fragments from chicken egg yolk immunoglobulin Y(IgY). J Immunol Methods 1993; 162: 155-64). The purified IgY antibodieswere dissolved in 5 ml of TBS containing 0.05% sodium azide and storedat −20° C.

Construction of scFv Antibody Libraries and Panning

The antibody libraries were established based on the previous report(Andris-Widhopf J, Rader C, Steinberger P et al. Methods for thegeneration of chicken monoclonal antibody fragments by phage display. JImmunol Methods 2000; 242: 159-81). Briefly, spleens harvested fromchickens following the final immunization were placed immediately inTrizol (Gibco BRL., USA) for homogenization. Ten μg of total RNA wasreversely transcribed into the first-strand cDNA using a SuperScript RTkit (Invitrogen, USA). After amplification using chicken-specificprimers, PCR products of heavy and light chain variable (VH and VL)regions were subjected to a second round of PCR to form full-length scFvfragments with a short or long linker, which were further digested withSfiI and cloned into the pComb3X vector. Recombinant phage DNAs weretransformed into E. coli ER2738 strain by electroporation (MicroPulserfrom Bio-Rad). The production of recombinant phages was initiated by theaddition of wild-type VCS-M13 helper phage, which were subsequentlyprecipitated with 4% polyethylene glycol 8000 and 3% NaCl (w/v), andfinally re-suspended in 1× phosphate-buffered saline (PBS) containing 1%bovine serum albumin (BSA). Then, 10¹¹ plaque-forming units (pfu) ofrecombinant phages in the scFv antibody libraries were added to wellspre-coated with purified His-CaENO1 protein (0.5 μg/well) and incubatedat 37° C. for 2 hrs. After the unbound phages were removed, bound phageswere eluted with 0.1 M HCl/glycine (pH 2.2)/0.1% BSA, neutralized with 2M Tris base buffer and used to infect the E. coli ER2738 strain. Theamplified phages were precipitated and recovered as described above forthe next round of selection. After 4th biopanning, total phagemid DNAfrom E. coli ER2738 was purified and transformed into E. coli TOP10F′. Apanel of randomly selected clones was cultured overnight, diluted 100×in super broth containing 1 mM MgCl2 and ampicillin (50 g/ml) andfurther grown for 8 hr. After induction with 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) overnight, the bacteria wereharvested through centrifugation, resuspended in histidine (His)-bindingbuffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH7.4), andlysed by 3 cycles of freezing, thawing/sonication. The scFv antibodieswere purified using Ni2⁺-charged Sepharose (GE Healthcare Bio-SciencesAB, Sweden) according to the manufacturer's instructions. The purifiedscFv antibodies were further concentrated in 1×PBS using Amicon Ultra-4Centrifugal Filter Devices (Merck Millipore, Germany) and examined fortheir binding or neutralizing capability against C. albicans.

C. albicans Growth and Hyphal Formation

To determine the effect of scFv antibodies on the cell growth and hyphalformation, anti-CaENO1 IgY (0.5 mg/ml) or CaS1 scFv (0.5 mg/ml) or PBSwere pre-incubated with C. albicans (1×10⁶ cfu) for 1 hr at 37° C. Afterincubation, 1 ul of each mixture in 10-fold dilutions was spotted on YPDagar plate and incubated at 37° C. overnight. 10³ cfu of C. albicans wasmixed with 0 (1×PBS control), 10 or 100 g/ml CaS1 scFv at roomtemperature for 1 hr. Thereafter, 1 ul of each mixture was spotted onYPD agar plate and incubated at 37° C. for 5 days.

Candida spp. Strains.

C. albicans (SC 5314), C. kruesi (clinical isolate), C. tropicalis (BCRC20520), C. parapsilosis (BCRC 20515) and C. glabrate (BCRC 20586) werekindly provided by Dr. Ching-Hua Su from Taipei Medical University,Taipei, Taiwan. C. albicans (CA6-17, CA7-26, CA7-3, CA10-50, CA7-30,CA10-65), C. tropicalis (CT11-52, CT6-29, CT6-50, CT12-54), C. glabrate(CG5-8, CG8-11, CG7-37, CG5-66), and C. parapsilosis (CP8-20, CP12-37,CP6-20, CP7-17, CP8-48) were kindly provided by Department of LaboratoryMedicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.Candida species were cultured in YPD medium and their identities wereconfirmed by CHROMagar Candida plate (CHROMagar, Paris, France). MICtest strips were used and MIC was read at 80% inhibition as recommendedby the manufacturer.

Western Blotting

To detect the presence of ENO1 proteins, the purified recombinant ENO1protein or cell lysates of five Candida spp were subjected to SDS-PAGEanalysis, transferred onto nitrocellulose membranes (AmershamBiosciences, UK) and then blocked with 5% skim milk in TBST for 1 hr.The anti-CaENO1 IgY from chicken after 7^(th) immunization (1:3,000) orpurified CaS1 scFv antibody (1 μg/ml) were added and incubated for 1 hrat room temperature. After vigorous washings, horseradish peroxidase(HRP)-conjugated polyclonal donkey anti-chicken IgY antibodies (1:3,000)(Bethyl Laboratories, Montgomery, Tex., USA) were added and incubatedfor an additional 1 hr for detecting the bound IgY antibodies. However,goat anti-chicken light chain antibodies (1:3,000) (Bethyl Laboratories,Montgomery, Tex., USA), followed by HRP-conjugated donkey anti-goat IgGantibodies (Jackson ImmunoResearch, USA) were used for detecting thebound scFv antibodies. After washings as above, the membranes weredeveloped with diaminobenzidine (DAB) or ECL substrate. The ImageQuantLAS4500 was used for ECL intensity detection.

ELISA and Competitive ELISA

To examine their binding reactivity, a series of diluted IgY antibodies(500-256,000-fold) purified from chicken after 7^(th) immunization orthe recombinant CaS1 scFv antibodies (40-0.078 μg/ml) were incubatedwith the purified CaENO1 (10 μg/ml) immobilized on ELISA plate wells.After vigorous washings, the bound IgY antibodies were detected byadding HRP-conjugated polyclonal donkey anti-chicken IgY antibodies(1:3,000) (Bethyl Laboratories, Montgomery, Tex., USA) while the boundCaS1 scFv antibodies were detected by goat anti-chicken light chainantibodies (1:3,000) (Bethyl Laboratories, Montgomery, Tex., USA),followed by HRP-conjugated donkey anti-goat IgG antibodies (JacksonImmunoResearch, USA). After washing as above, a tetramethylbenzidine(TMB) substrate solution (Sigma, USA) was added to the wells for colordevelopment. The reaction was stopped with 1 N HCl and optical densitywas measured at 450 nm using an ELISA plate reader (BioTek Synergy HT).Dissociate constant (K_(D)) was calculated according to the equation,grams/molecular weight (Da)×volume (L)⁻¹.

For the competitive ELISA, the procedure described above was performedexcept a series of diluted CaENO1 proteins (50-0.097 μg/ml) were firstmixed with equal volume of CaS1 scFv antibodies (1 μg/ml) for 1 hr andadded to the plates for detecting the binding specificity. The ELISAtests were carried out in the duplicated wells for each sample. ELISAdata were presented as mean±SD of the duplicated experiments.

Fibrin Matrix-Gel Degradation Analysis

The matrix gel was prepared for fibrinolysis activity detection³³ . C.albicans (10⁶ cells) were washed and incubated with or without CaS1scFv(10 and 100 μg) at 37° C. for 1 hr. After incubation, cells were washedand incubated with plasminogen (10 μg) for 30 min. The mixtures werewashed with PBS to remove free plasminogen. The resulting cell pelletswere placed in a matrix gel that contained 1.25% low-melting-temperatureagarose, thrombin (0.05 U/ml, Sigma) and fibrinogen (2 mg/ml, Sigma).The gel was incubated in a humidified chamber at 37° C. for 10-14 hrsuntil the appearance of clear spots indicated the presence offibrinolysis activity.

Adhesion Assay

Cell adhesion assay was performed using human oral epidermal cells(OECM-1) (XXX). C. albicans (1×10⁶ cfu) were pre-incubated with CaS1scFv antibodies (50 or 100 μg) at 37° C. for 1 hr. After incubation, themixtures were two-fold diluted and added onto 10⁴ OECM-1 cells culturedin 96-well plates. The plates were further incubated at 4° C. for 2 hrs.After being washed three times with 1×PBS, the plates were fixed with10% formaldehyde. After being washed as above, the wells were blockedwith skim milk for 1 hr. Thereafter, rabbit anti-C. albicans antibodies(1:3,000, Bethyl, USA) were added and incubated for another 1 hr,followed by adding the HRP-conjugated anti-rabbit antibodies (1:3,000,Bethyl, USA). Washings as above were always carried out between steps.TMB was used for color development, which was stopped with 1 N HCl. Theintensity of the color was measured at 450 nm on an ELISA plate reader.

Mouse Model of C. albicans Infection

A total of 20 ICR female mice (purchased from National Laboratory AnimalCenter, Taiwan) weighing about 30 g were randomly grouped with 5 mice ineach group. C. albicans were grown overnight and washed in normalsaline. Four groups of mice were treated through the lateral tail veinwith the following preparations: (i) 1×PBS alone; (ii) 1×10⁶ cfu of C.albicans cells pre-incubated with 50 μg of CaS1; (iii) 1×10⁶ cfu of C.albicans cells pre-incubated with 100 μg of CaS1; (iv) 1×10⁶ cfu of C.albicans cells pre-incubated with anti-CaENO1 IgY. Mice were monitoredfor survival at 1 day intervals for 10 days. All treatments and handlingof mice were carried out according to animal experimental protocolsapproved by the Institutional Animal Care and Use Committee of TaipeiMedical University.

Example 1 Expression and Purification of Fusion Protein of his-C.albicans Alpha-Enoloase (his-CaENO1)

C. albicans alpha-enolase genes were constructed in pQE30 plasmids toform pQE30-CaENO1 vector and then the resulting vectors were transformedwith E. coli BL21 cells. The expression of the cells were was inducedwith IPTG at a concentrations 1.0 mM, 0.5 mM and 0.1 mM for 4 hours, 5hours, 8 hours and overnight. It was found that the expressed proteinhads a molecular weight at of about 49 kDa by SDS-PAGE (FIG. 1A) andWestern blot analysis (FIG. 1B).

C. albicans alpha-enolase genes were constructed in pQE30 plasmids toform pQE30-CaENO1 vector and then the resulting vectors were transformedwith E. coli BL21 cells. The expression of the cells were was inducedwith IPTG at a concentrations 1.0 mM, 0.5 mM and 0.1 mM for 4 hours, 5hours, 8 hours and overnight. It was found that the expressed proteinhads a molecular weight at of about 49 kDa by SDS-PAGE and Western blotanalysis. To confirm that the expressed protein mentioned above is thedesired CaENO1 protein, the protein obtained in Example 1 was purifiedwith Ni SepharoseTHigh Performance, the purified protein was analyzed bySDS-PAGE and a protein with 49 kDa was confirmed as His-CaENO1 (FIG.1C).

Example 2 Binding Assay of his-CaENO1 IgY Polyclonal Antibody to Antigenhis-CaENO1

50 μg of the purified His-CaENO1 protein of Example 1 was administeredto chickens to produce anti-his-CaENO1 IgY polyclonal antibodies. Thepolyclonal antibodies were purified from eggs by SDS-PAGE and Westernblot analysis. The binding ability of the purified antibodies wasassayed by Western blot and enzyme-linked immunosorbent assay (ELISA).In the ELISA assay, BSA was used as negative control, mouse anti-his IgGwas used as the first antibody and HRP rabbit anti-mouse IgG was used asthe second antibody. After the Western blot analysis, anti-his-CaENO1IgY polyclonal antibodies specifically binding to His-CaENO1 wereproduced after immunization three times and its binding abilityincreased with each round of immunization.

Example 3 Binding Activity of Chicken Anti-CaENO1 IgY, Anti-CaENO1Library Construction and Panning

The humoral immune response of chicken anti-CaENO1 IgY was identified byELISA and Western blot (FIG. 2 B). As compared to pre-immune serum andun-related BSA protein, IgY after 7^(th)-immunization (from500-256000-fold dilution) recognized CaENO1. (FIG. 2 A)

Two libraries with short linker and long linker were constructed asshown in Table 1. The sizes for short and long linker libraries wereestimated to be 2.4×10⁶ and 1.36×10⁷, respectively. The elution titerafter each panning were shown (Table 1). After four rounds of panning,the CaENO1 binding phage variants were greatly enriched. These resultssuggested the non-specific binding phage was removed throughout thepanning process and the clones with specific binding affinity wereenriched. The sequences were confirmed to belong to chickenimmunoglobulin germline gene. The short linker with highest bindingactivity to CaENO1 was identified. This anti-CaENO1 scFv monoclonalantibody was named CaS1.

TABLE 1The anti-CaENO1 library size and eluted phage titers after each round ofpanning. Linker Library Eluted phage titers after each round of panningLibrary length* size 1^(st) 2^(nd) 3^(rd) 4^(th) CaENO1-S  7 aa 2.4 x 10⁶  9.6 x 10⁴ 9.0 x 10⁵  7.2 x 10⁵ 3.0 x 10⁶ CaENO1-L 18 aa1.36 x 10⁷ 2.75 x 10⁵ 1.2 x 10⁶ 1.32 x 10⁶ 1.2 x 10⁵ *Linker length of 7aa and 18 aa are GQSSRSS (SEQ ID NO: 17) and GQSSRSSGGGGSSGGGGS (SEQ IDNO: 18), respectively.

Example 3 Gene Sequencing of scFv Antibody

The antibody purified from the chicken includes framework region (FR)and complementarity determining regions (CDRs). Colonies #1S1 to #1S12were isolated and sequenced using ompseq primers(5′-AAGACAGCTATCGCGATTGCAGTG-3′, SEQ ID NO: 29), and the sequencingresults were analyzed by BioEdit software and then the resultingsequences were compared with chicken germline. It was found that theantibodies in the ten clones have the same sequences in light chain andheavy chain (FIG. 3).

Total phagemid DNA from the last biopanning was transformed into TOP10F′E. coli to analyze individual scFv. The colony containing scFv genefragment was selected and cultured in 10 mL LB (Lauria-Bertani) broth(50 μg/mL Ampicillin) with shaking at 37° C. overnight; then, theculture was transferred to another 100 mL LB (with 50 μg/mL Ampicillin)until OD₆₀₀ reached between 0.4 to 0.8. Thereafter, the resultingculture was incubated with 0.5 mM IPTG for 6-8 hours to express theHis-tagged CaS1 scFv protein. Then, the culture was subjected tocentrifugation and the supernatant was discarded, and the pellet wasresuspended with 1 mL of His-binding buffer (20 mM sodium phosphate, 0.5M NaCl, 20 mM imidazole, pH 7.4). The E. coli cells were disrupted withsonication; then, the sample was subjected to 3000 g centrifugation for5 minutes. Next, the CaS1 scFv fusion protein in the supernatant waspurified by Ni Sepharose™ High Performance (GE healthcare Life science,USA) as suggested by the manufacturer. In brief, the sample supernatantwas added to the Sepharose, mixed for 1 hour and subjected to 1000 gcentrifugation for 5 minutes. The supernatant was discarded. TheSepharose was washed with 1 mL His-binding buffer 2 times to get wash 1and wash 2. 500 μL of His-elution buffer was added to the Sepharose andmixed for 1 hour. The Sepharose was subjected to 1000 g centrifugationfor 5 minutes and the supernatant (the elution 1) was collected. Theabove steps were repeated to get elution 2. Elutions 1 and 2 contain thepurified His-CaS1 scFv. Finally, the Sepharose was resuspended in 50 μLof His-binding buffer. Fractions of binding supernatant, wash 1, wash 2,elution 1, elution 2 and the Sepharose were analyzed by 12% SDS PAGE(FIG. 4).

Example 5 K_(D) Determination of CaS1 scFv by ELISA and CompetitiveELISA

0.25 μg of His-CaENO1 was added to each well of a 96 half area plate,and the plate was incubated at 37° C. for 1 hour to allow the adsorptionof the proteins to the bottom of the wells. The proteins were discardedand 5% skimmed milk was added to the wells, and the plate was incubatedat 37° C. for 1 hour for blocking. Meanwhile, 1 μg/mL CaS1 was incubatedwith each 2-fold and 10 times serial diluted samples from 50 μg/mL freeform His-CaENO1 (i.e., 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.13μg/mL, 1.56 μg/mL, 0.78 μg/mL, 0.39 μg/mL, 0.19 μg/mL of free formHis-CaENO1) at room temperature for 1 hour. Then, these samples wereadded to the coated and blocked wells described above and incubated at37° C. for 1 hour for conducting a competition reaction. Thereafter, thewells were washed with PBST 6 times; then, goat anti-chicken light chain(1:3000 diluted) was added thereto and incubated at 37° C. for 1 hour.Thereafter, the wells were washed with PBST 6 times; then donkeyanti-goat HRP antibody (1:5000 diluted) was added thereto and incubatedat 37° C. for 1 hour. Thereafter, the wells were washed with PBST 6times; then, the color reaction was initiated by adding3,3′,5,5′-Tetramethylbenzidine (TMB), and the reaction was terminated by1N HCl. The absorbance at 450 nm wavelength was detected to get OD₄₅₀.To determine the binding activity of CaS1 to CaENO1, ELISA (FIG. 5A-B)and competitive ELISA (FIG. 5C-D) were performed. As seen in FIGS. 5Aand 5B, the binding activity of CaS1 scFv antibody to CaENO1 wereconcentration dependent, and the K_(D) are 1.88×10⁻⁸ M and 8.9×10⁻⁸ M,respectively as measured by ELISA and competitive ELISA.

Example 6 CaS1 scFv Binding to Cell Lysate of C. albicans and C.tropicalis

This CaS1 scFv was evaluated for its binding activity to differentspecies of Candida. Five common strains of Candida were obtained, totallysate of five species Candida were analyzed on SDS-PAGE (FIG. 6A left)and subsequent Western blot. As seen in FIG. 4A middle, anti-CaENO1polyclonal IgY are able to recognize five species of Candida tested asexpected. However, CaS1 scFv can only reacted with C. albicans as seenin FIG. 6A right lane 1 and C. tropicalis as seen in lane 3. This CaS1scFv cannot react with C. krusei (lane 2), C. parapsilosis (lane 4) andC. glabrate (lane 5). The ENO1 protein sequence between C. albicans andC. tropicalis are 83% homology, therefore, the reason that CaS1 canrecognized C. albicans as well as C. tropicalis may suggest these twospecies share similar epitope which can bind to CaS1 scFv.

Furthermore, we exame the CaS1 scFv binding activity to fluconazoleresistance and suspcetible stains of Candida spp. from clinic. TheCandida spp. and their MIC were listed in Table 2. As shown in FIG.4D-G, CaS1 scFv can bind to fluconazole resistance and susceptible C.albicans (FIG. 6B) and C. tropicalis (FIG. 6C) but not to C. glabrateand C. parapsilosis (data not shown). These data suggesting CaS1 scFvcan be potentially used for the diagnosis and/or treatment of thefluconazole resistance C. albicans and C. tropicalis.

In addition, we also observed that CaS1 scFv can bind to differentspecies of ENO1 as seen in FIG. 6D. ENO1 for C. albicans (CaENO1), S.pneumoniae (SpENO1), S. aureus (SaENO1), mouse (mENO1) and human (hENO1)were purified and subjected on SDS-PAGE (FIG. 6D left) and Western blot(FIG. 6D right). CaS1 scFv bind not only CaENO1 but also SpENO1 andSaENO1, but very little to mENO1 and hENO1 (FIG. 6D right). Theseresults suggest that ENO1 from C. albicans, S. pneumoniae and S. aureus,may share similar epitope which can bind to CaS1 scFv.

TABLE 2 Clinical fluconazole resistance and susceptible Candida spp. andtheir MIC. Specimen organism drug MIC (□g/ml) Interpretation* CA6-17 C.albicans fluconazole 16 R CA7-26 C. albicans fluconazole 8 R CA7-3 C.albicans fluconazole 2 S CA10-50 C. albicans fluconazole 2 S CA7-30 C.albicans fluconazole 1 S CA10-65 C. albicans fluconazole 1 S CT6-29 C.tropicalis fluconazole 32 R CT11-52 C. tropicalis fluconazole 32 RCT6-50 C. tropicalis fluconazole 8 R CT12-54 C. tropicalis fluconazole 8R CG5-8 C. glabrate fluconazole 64 R CG8-11 C. glabrate fluconazole 64 RCG7-37 C. glabrate fluconazole 32 S CG5-66 C. glabrate fluconazole 16 SCP8-20 C. parapsilosis fluconazole 256 R CP12-37 C. parapsilosisfluconazole 256 R CP6-20 C. parapsilosis fluconazole 16 R CP7-17 C.parapsilosis fluconazole 8 S CP8-48 C. parapsilosis fluconazole 4 S *Rfor resistance; S for susceptible.

Example 7 Flow Cytometry Assay for Identification of CaS1 Antibody toAlpha-Enolase on Cell Surface of C. Albicans

Anti-CaENO1 IgY, scFv CaS1 and control scFv were contacted with C.albicans and then subjected to flow cytometry assay. Anti-CaENO1 IgY,scFv CaS1 and control scFv show 89.7+/−2.8%, 46.64+/−3.1% and 23+/−2.3%FITC fluorescence reaction (FIG. 7A), representing that the antibody canbind to alpha-enolase on the surface of C. albicans. Moreover, propidiumiodide for dying was used to identify the mortality rate of C. albicans,and it was found that Anti-CaENO1 IgY, scFv CaS1 and control scFv kill90+/−1.5%, 37+/−0.8%, 21+/−1% of C. albicans (FIG. 7A). As shown in FIG.7B, in comparison with control svFv and Anti-CaENO1 IgY, scFv CaS1 canbind to the alpha-enolase on the surface of C. albicans and thus killthem.

Example 8 Immunofluorescence Assay for Identification of CaS1 Antibodyto Alpha-Enolase on Cell Surface of C. Albicans

Anti-CaENO1 IgY, scFv CaS1 and control scFv were used inimmunofluorescence assay for identification of CaS1 antibody toalpha-enolase on cell surface of C. albicans. As shown in FIG. 8,anti-CaENO1 IgY (1) and scFv CaS1 (2) bind to the alpha-enolase on thesurface of C. albicans, whereas the control scFv (3) does not show thebinding.

Example 9 Attenuation of C. albicans Growth and Hyphal Formation by CaS1scFv

We evaluated the influence of CaS1 on growth of C. albicans, wepre-incubated CaS1 scFv with C. albicans and plated on the agar. As seenin FIG. 9A, CaS1 scFv decreased C. albicans growth as observed on YPDagar plate when diluted to the 10⁻⁴ as compared to control. Theinhibitory effects for anti-CaENO1 IgY is as good as scFv CaS1 scFv.

Hyphal formation has also been shown to play a critical role in thevirulence of the C. albicans. So we further investigated whether CaS1scFv affects the hyphal formation. We pre-incubated CaS1 scFv with C.albicans and the colony morphology was examined. As seen in Figure. 9B,along the edge of the control colony, hyphae were clearly visible,whereas very little or none were observed for the CaS1 scFv treatedcolonies. The attenuation effect for hyphae formation is obvious whenCaS1 scFv were pre-incubated with C. albicans.

Example 10 Inhibition of Binding of C. Albicans to Human OralKeratinocyte OECM-1 Cells

C. albicans cells (1×10⁶) were treated with 50 μg and 100 μg of scFvCaS1, respectively, and the resulting mixtures were added to human oralkeratinocyte OECM-1 cells to test the binding ability of C. albicanscells to OECM-1 cells. In comparison with the PBS control, the 50 μg and100 μg of scFv CaS1 significantly reduced the binding of C. albicanscells to OECM-1 cells (FIG. 10).

Example 11 Effect of CaS1 scFv on the Binding of ENO1 to Plasminogen

It is well known that surface ENO1 act as a plasminogen receptor³²,binding to plasminogen will activate it to plasmin and leading to thedegradation of the fibrinogen (extracellular matrix) containing in thegel. We performed matrix-gel studies to test the possible biologicalsignificance of CaENO1-plasminogen association and the effect of CaS1scFv on this binding was observed.

A representative plate was seen in FIG. 11. In the absence ofplasminogen, Candida alone (FIG. 11-1) or CaS1 scFv alone (FIG. 9-6)showed no fibrinolysis activity. C. albicans incubated with 1 and 10 μgof plasminogen, resulted in a significant increase in fibrinolyticactivity (FIGS. 11-2 and 11-3). This result suggests that CaENO1 boundplasminogen can be activated by thrombin, present in the matrix gel, todigest the surrounding fibrinogen. However, fibrinolytic activities canbe significantly inhibited by pre-incubated C. albicans with CaS1 (10and 100 μg) as compared to in the presence of plasminogen alone as showin FIGS. 11-4 and 11-5. The inhibition of the degradation of thefibrinogen in the gel is obvious. Our results suggest that binding ofCaENO1 to plasminogen was significantly reduced by CaS1 scFv antibodiesin a dose-dependent manner.

Example 12 Neutralization of C. Albicans Toxicity by ScFv CaS1 to ExtendSurvival Rate of Infected Mice

1×10⁶ cells of C. albicans solutions were mixed with 100 μg of each ofthe anti-CaENO1 IgY, scFv CaS1 and control scFv and then injected intoICR mice (5 mice for each group). After 10 days, the survival rates ofthe anti-CaENO1 IgY, scFv CaS1 and control scFv groups were 100%, 80%and 0%, respectively. It was found that the anti-CaENO1 IgY and scFvCaS1 can neutralize the toxicity of C. albicans and thus can extend thelife of mice or protect the mice from death.

Example 13 Humanization of CaS1 scFv by CDR Grafting

Two humanized CaS1 scFv (V1 and V3) were designed. Humanized CaS1 scFvV1 was grafted onto human framework (Protein Data Bank: 2JIX-L, 2ZKH-H),this most suitable length sequence was analyzed by Discovery Studiosoftware. Humanized CaS1 scFv V3 was grafted onto human framework thatthe sequence of Avastin® (Protein Data Bank: 2FJG). These humanized CaS1scFv (V1 and V3) were synthesized by Gemonics BioSci & Tech (New TaipeiCity, Taiwan), cloned into pComb3X vector, and transferred into Top 10E. coli for protein expression. The expressed V1 and V3 proteins werepurified by Ni⁺ sepharose and analyzed by SDS-PAGE and Western blot.

As the results shown in FIG. 12, the humanized CaS1 scFv V1 (FIG. 13A)and V3 (FIG. 13B) antibodies were designed to synthesize a plasmid DNAwith the target gene, and the DNA was then transferred into Top 10 E.coli for expression. Then, the expressed protein was purified with Ni⁺Sepharose, and analyzed by SDS-PAGE. Afterward, the expressed proteinwas confirmed by Western blot (anti-HA tag).

Example 14 Determination Binding Ability of CaENO1 with hzCaS1 V1 and V3

The hzCaS1 V1, V3 and CaS1 scFv were used to recognize recombinantCaENO1 protein. As shown in FIG. 13, (A) the Coomassie blue was used tostain; (B) the mouse anti-human κ, λ IgG and the HRP conjugated rabbitanti-mouse IgG was used in the Western blot (right panel); (C) the mouseanti-HA IgG and HRP conjugated rabbit anti-mouse IgG was used; and (D)The goat anti-chicken light chain IgG and HRP conjugated donkeyanti-goat igG was used. Lane 1: hzCaS1 scFv V1. Lane 2: hzCaS1 scFv V3.Lane 3: CaS1 scFv.

Example 15 K_(D) Determination of hzCaS1 V1 and V3 scFv by ELISA

Purified hzCaS1 V1 and V3 scFv were used to recognize recombinant CaENO1protein. The hzCaS1 V1 and V3 scFv were used as primary antibody withseries dilution. The goat anti-chicken light chain IgG was used assecondary antibody. The HRP conjugated donkey anti-goat IgG was used todecet (see FIG. 14 (A) and (C)). OD value was calculated intopercentage. The K_(D) or 50% effective concentration (EC₅₀) of scFv werecalculated and expressed by molarity (M). K_(D) of hzCaS1 V1 and V3 scFvis 1.51 ug/ml=4.6×10⁻⁸ M and 2.12 ug/ml=8.4×10⁻⁸ M, respectivity. ELISAdata were represented as mean±SD of the duplicated well (see FIG. 14 (B)and (D)).

Example 16 CaS1, hzCaS1 V1 and V3 scFv Inhibits CaENO1 Binding toPlasminogen

CaENO1 on Ni⁺ sepharose was treated with 100 ug hzCaS1 V1, V3 and CaS1scFv for 1 hour, following incubating with plasminogen (20 ug) for 1hour. Each experimental group of CaENO1 with CaS1 scFv were dropped ontogel and incubated at room temperature for 2 days to observe gel degraderesult an the results are shown in FIG. 15. FIG. 15-1 in the presence ofplasminogen (1 ug/ul) alone (positive control) show a significantfibrinolytic activity as seen in the gel. FIG. 15-2 CaENO1 on NiSepharose™ (10 ug) alone (negative control) with no plasminogen show nofibrinolysis activity in the gel. FIG. 15-3 CaENO1 on Sepharose™ (10 ug)with plasminogen (20 ug) show fibrinolytic activity. FIG. 15-4,5,6CaENO1 on Sepharose™ (10 ug) treated with hzCaS1 V1 (FIG. 15-4), V3(FIG. 15-5), CaS1 scFv (FIG. 15-6) (100 ug) and incubated withplasminogen (20 ug) show no fibrinolytic activity as compared to 15-3.Therefore, result suggests that CaENO1 bound plasminogen can beactivated by thrombin, present in the matrix gel, to digest thesurrounding fibrinogen. However, fibrinolytic activities can besignificantly inhibited by pre-incubated CaENO1 with hzCaS1 V1, V3, CaS1scFv as compared to in the presence of plasminogen as show in FIG. 15-3.The inhibition of the degradation of the fibrinogen in the gel isobvious (FIG. 15-4,5,6). Our results suggest that binding of CaENO1 toplasminogen was significantly reduced by hzCaS1 V1, V3, CaS1 scFvantibodies.

Example 17 Epitope Mapping of CaENO1 Using CaS1 scFv

Nine PCR-amplified fragments were obtained using full-length CaENO1(1323 bp) as template, ligated into PET-21a vector and transformed intoBL-21 E. coli. The identity of inserted fragments was confirmed bysequencing (Genomics BioSci & Tech, New Taipei City, Taiwan). Proteinexpression was carried out by IPTG induction. SDS-PAGE and Western blotwere used to characterize the expressed recombinant proteins.

For epitope mapping of CaENO1, the purified CaS1 scFv was used torecognize recombinant CaENO1 protein on Western blot and ELISA. Theepitope region was mapped to contain 198 bp nucleotides, which deducedamino acid sequences (residues 235 to 300) areDKAGYKGKVGIAMDVASSEFYKDGKYDLDFKNPESDPSKWLSGPQLADLYEQLISEYPIVS IEDPF (SEQID NO:19) (66 amino acids).

To further determine the epitopic location, site directed mutagenesis(Kunkel method) was used to construct nine peptide-expressing phagesaccording to the nucleotide sequences of 198 bp of mapped antigenicfragment above mentioned with minor modification (“Chapter 2,Constructing phage display libraries by oligonucleotide-directedmutagenesis” on Phage Display-A Practical Approach, edited by TimClackson and Henry B. Lowman, OXFORD University Press 2004). Themodified pCANTAB 5E DNAs carrying nucleotide sequences coding theantigenic epitopes were transformed into ER2738 E. coli. The phagemidDNAs were extracted from E. coli and analyzed for insertion with EcoRIenzyme restriction. Each resulting phage expressing 10 amino acids onviral particle surface was examined for their reactivity against CaS1scFv antibody on ELISA. The combined results showed CaS1 scFv recognizedthe amino acid residues on antigenic epitopes located among₂₄₀KGKVGIAMDV₂₄₉ (SEQ ID NO:3) and ₂₇₈PQLADLYEQLISEYP₂₉₂ (SEQ ID NO:4).

Nine recombinant CaENO1 proteins as above were used to recognize thepurified plasminogen on dot blot. The results showed CaS1 scFv antibodybinds to a fragment of plasminogen spanning amino acid residues 301 to437, which sequences areAEDDWDAWVHFFERVGDKIQIVGDDLTVTNPTRIKTAIEKKAANALLLKVNQIGTLTESIQAANDSYAAGWGVMVSHRSGETEDTFIADLSVGLRSGQIKTGAPARSERLAKLNQILRIEEELGSEAIYAGKDFQKA (SEQ ID NO:20). The above results are shown in FIG. 16.

Example 18 CaS1 scFv Prolongs the Survival Rate of ICR Mice Infectedwith C. albicans

We evaluated the survival rate of the CaS1 on animal model. InitiallyICR mice were tail-vein injected with 1×10⁶ cells of C. albicans withPBS alone or cells pre-incubated with CaS1 (10 and 100 μg) for one hourat 37° C. As seen in FIG. 17, pre-incubated with CaS1 (10 and 100 Gg)pro-long the survival rate of mice to 40% and 80% respectively, ascompared to PBS control. Anti-Deinagkistrodon acutus scFv (anti-DA) isan antibody to snake venom, which was used as an unrelated scFv control.These data suggest CaS1 scFv provide partial protect activity againstlethal challenge of candidemia on ICR mice.

Example 19 Candida Albicans Biofilm Formation Inhibition Assay

Candida albicans cells were cultured in YPD medium and then collected.The cells were washed with PBS and resuspended in YPD medium containing100 mM glucose at a cell density of 10⁷ cells/ml. A 96-well plate wastreated with CaS1 scFv or fluconazole and then inoculated with 1 ml ofthe cell suspension. After a two-hour adhesion period, the inoculum wasremoved by washing with PBS, and YPD medium containing 100 mM glucosewas applied to the plate. The plate was treated with CaS1 scFv orfluconazole and biofilms were grown for 72 hours at 37° C. The plate waswashed with PBS, and then biofilm formation was observed by microscopy(FIG. 18).

What is claimed is:
 1. An isolated anti-CaENO1 antibody or anantigen-binding portion thereof, comprising a light chaincomplementarity determining region 1 (L-CDR1) of SEQ ID NO:5; a lightchain CDR2 (L-CDR2) of SEQ ID NO:6; and a light chain CDR3 (L-CDR3) ofSEQ ID NO:7; and a heavy chain CDR1 (H-CDR1) of SEQ ID NO:8; a heavychain CDR2 (H-CDR2) of SEQ ID NO:9; and a heavy chain CDR3 (H-CDR3) ofSEQ ID NO:10; such that said isolated antibody or antigen-bindingportion thereof binds to CaENO1.
 2. The antibody of claim 1, wherein theisolated anti-CaENO1 antibody is a monoclonal antibody, chimericantibody or humanized antibody.
 3. The antibody of claim 1, wherein thelight chain of the antibody comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:13 and 14; or wherein the heavychain of the antibody comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 15 and
 16. 4. The antibody of claim 1,which is a humanized antibody comprising (i) a light chain having anamino acid sequence selected from the group consisting of SEQ ID NOs:13and 14, and (ii) a heavy chain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:15 and
 16. 5. The humanizedantibody of claim 4, which comprises: (i) a light chain having an aminoacid sequence of SEQ ID NO:13 and (ii) a heavy chain having an aminoacid sequence of SEQ ID NO: 15 or (iii) a light chain having an aminoacid sequence of SEQ ID NO: 14 and (iv) a heavy chain having an aminoacid sequence of SEQ ID NO: 16.